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Nan Wang,1,2 Hongshan Ge,1 Shu Zhou1 1Department of Obstetrics and Gynecology, Taizhou People’s Hospital, Taizhou, Jiangsu Province, People’s Republic of China; 2Department of Graduate College, Dalian Medical University, Dalian, Liaoning Province, People’s Republic of ChinaCorrespondence: Hongshan Ge Email dafeng76@126.comBackground: The World Health Organization (WHO) has defined Unexplained Recurrent Spontaneous Abortion (URSA) as three and more consecutive miscarriages before the 20th week of gestation. To date, empiric therapy for patients with unexplained recurrent pregnancy loss (URPL) is not precise. Studies have shown that URSAs are associated with Th1/Th2 and Th17/Treg immune imbalances at the maternal-fetal interface. The immunosuppressant cyclosporine A (CsA) is widely used in patients with organ transplantation or autoimmune diseases, and it has a good safety profile in pregnant women. However, high-quality evidence for CsA treatment of URSAs is lacking. Our purpose with this study is to evaluate the efficacy and safety of CsA for improving pregnancy outcomes in patients with URSAs and to explore the role of CsA in regulating the immune balance.Methods/Design: We expect to officially initiate our study at the Taizhou People’s Hospital in March 2022. We defined the live birth rate as the primary outcome, and the secondary outcomes include the rates of successful pregnancy, miscarriage, pregnancy complications, and adverse pregnancy outcomes, and newborn birth weights. Patients who meet URSA eligibility criteria will be randomized in a 1:1 ratio into either a study group receiving CsA 2 weeks after fertilization or a control group receiving placebo at 2 weeks after fertilization (the women in both groups will receive the relevant treatment for 6 months). In addition, we will collect peripheral blood samples of the participants before and after the treatments, and we will isolate mononuclear cells and measure cytokine levels (IFN-γ, TNF-α, IL-2, IL-10, IL-6, IL-4) and Th1/Th2, Th17/Treg ratios.Discussion: This is the first randomized controlled trial to evaluate the clinical and immunomodulatory effects of CsA on the pregnancy outcomes of women with URSA and our results will provide evidence to evaluate the use of CsA as a treatment for women with URSAs.Keywords: cyclosporine A, live birth rate, recurrent abortion of unknown cause, Th1, Th2

Final Thesis Curriculum in Veterinary Medicine Keratoconjunctivitis sicca (KCS) is a common ocular disease seen in dogs, and the management of the disease may prove to be challenging. The aim of this study was to analyse clinical cases of KCS in the Small Animal Clinic of Estonian University of Life Sciences regarding the breed, age, and sex of the affected dogs, the analysis of the diagnostic methods and treatment options utilised, and finally making recommendations for the management of dogs with KCS. The study data was collected from the Small Animal Clinic of Estonian University of Life Sciences patient database. The patient population consisted of 45 canines which were treated for KCS at the clinic from 2017-2022. There were 23 different breeds of dogs included, with Yorkshire terrier being most frequent (17.8%). There was no relationship found between the age groups (young, middle, and senior age; p = 0.989), sex (p = 0.837) and the summed Schirmer tear test (STT) results from both eyes. 60% of the study population was affected by KCS bilaterally and in these patients the summed STT result from both eyes was significantly lower than in unilaterally affected patients (p = 0.011 and p = 0.002 in left and right affected eye respectively). The STT results increased significantly from the initial visit to the second control in both eyes (left eye p = 0.013, right eye p = 0.002), with STT increasing more in the left than in the right eye. According to the findings, the most commonly applied treatment of eye cleansing, artificial tear replacers and topical cyclosporine A (CsA) seem to be effective in the management of KCS in the majority of the dogs in this study population. Kuiv keratokonjunktiviit (KCS) on koertel sageli esinev silmahaigus ja selle haiguse ravi võib osutuda väljakutseks. Käesoleva töö eesmärgiks oli analüüsida KCS kliinilisi juhtumeid Eesti Maaülikooli Väikeloomakliinikus haigestunud koerte tõu, vanuse ja soo osas, analüüsida kasutatud diagnostika meetodeid ja ravivõimalusi ning koostada soovitused KCS-ga koerte raviks. Uuringu andmed koguti Eesti Maaülikooli Väikeloomakliiniku patsientide andmebaasist. Patsientide populatsioon koosnes 45 koerast, keda raviti kliinikus KCS-a tõttu aastatel 2017–2022. Uuringus oli 23 erinevat tõugu koera, kusjuures Yorkshire'i terjer oli sagedamini esindatud (17.8%). Koerte vanuse kategooria (noor, keskmine vanus ja vanem; p = 0.989), soo (p = 0.837) ning summaarse Schirmeri pisaratesti (STT) tulemuste vahel seost ei leitud. KCS esines kahepoolselt 60% uuringupopulatsioonist ja nendel patsientidel oli mõlema silma summaarne STT tulemus oluliselt madalam kui ühepoolselt tabandunud patsientidel (p = 0.011 ja p = 0.002 vasakus ja paremas silmas). STT tulemused suurenesid oluliselt järgmisel kontrollil (vasak silm p = 0.013, parem silm p = 0.002), võrreldes esialgse visiidiga mõlemas silmas, kusjuures STT suurenes vasakus silmas rohkem kui paremas silmas. Tulemuste kohaselt, KCS-i ravis enamikul selle uuringupopulatsiooni koertel osutus tõhusamaks kõige sagedamini kasutatav ravi silmapuhastusega, pisaraasendajatega ja paikse tsüklosporiiniga (CsA).

Vernal keratoconjunctivitis (VKC) is a severe form of ocular allergy that compromises the quality of life of affected patients. Topical cyclosporine 0.1% cationic emulsion (CsA-CE) has been recently authorized for the treatment of severe VKC. We treated 29 VKC patients with on-label CsA-CE and recorded signs and symptoms, subjective patient's treatment satisfaction and the additional use of topical corticosteroids in case of exacerbations. CsA-CE was effective in reducing signs and symptoms in daily clinical practice. The overall subjective improvement of symptoms, efficacy of the treatment, tolerability to the drug and compliance reached a high level of subjective satisfaction score.55% of treated patients required the additional use of a 3-day course of topical dexamethasone with 1.13 ± 0.81 mean courses/month. In conclusions, VKC patients reported an overall high satisfaction with used the on-label topical CsA-CE with a limited use of additional topical corticosteroid treatment.

Objective: Cyclosporine A (CsA) and tacrolimus (Tac) are immunosuppressive drugs which is frequently used in organ transplantation. CsA can cause various side effects including gingival overgrowth (GO) and osteopenia. Tac has similar side effects to CsA but with different incidences. The aim of the present study was to evaluate and compare the effects of CsA and Tac on GO and alveolar bone resorption in rats. Methods: Sixty mature male rats were randomly and equally allocated into six groups, as follows: Control-I, Control-II, CsA-I, CsA-II, Tac-I and Tac-II. The Control-I and Control-II groups received, subcutaneously, 1 ml/kg 0.9% NaCl, while the CsA-I and CsA-II groups received 10 mg/kg CsA daily, and the Tac-I and Tac-II groups 1.5 mg/kg Tac daily. The Control-I, CsA-I and Tac-I groups were sacrificed on the 16th day and other groups on the 31st day. Histological and histomorphometric analysis of the buccal gingiva and tartrate-resistant acid phosphatase (TRAP) enzyme histochemistry of the alveolar bone were performed in the right mandibular segment of each animal. Results: GO was significantly greater in the groups administered CsA compared to the other groups (P0.05). CsA caused a significant increase in TRAP positivity (P0.05). Conclusions: Our results showed that Tac does not seem to cause GO and alveolar bone resorption. However, the deleterious side effects of Tac on the gingival tissues of rats may be time-related.

Cyclosporine A (CYA) is prescribed to prevent graft rejection after transplantation. Interferon-α (IFN-α), a natural cytokine, is prescribed for some types of malignancies, and hepatitis C virus. But both may cause neurologic complications such as depression following their chronic use. Melatonin is a hormone that helps regulate the circadian rhythm, additionally the melatonin system is connected to depression. The goal was evaluating the antidepressant effect of melatonin following IFN-α, and CYA administration in mice. Male NMRI mice (25-30 g) were used, IFN-α (1600000 IU/kg, sc), CYA (20 mg/kg, ip), melatonin (50 mg/kg, ip), and fluoxetine (20 mg/kg, ip) were administered daily. After evaluating the locomotor activity, depression was assessed by splash test, forced swimming test (FST), and the sucrose preference test. There was no significant difference in the locomotor activity amongest different animal groups. Follow- ing melatonin and IFN-α co-administration immobility time in FST decreased (58.50±19.4s, p

We herein report the case of 21-year-old female diagnosed with adult-onset Still's disease (AOSD) three years earlier who presented with fever and right upper abdominal pain. She was diagnosed with acute acalculous cholecystitis (AAC) based on hepatic dysfunction, elevated C-reactive protein, and gallbladder wall thickening on abdominal ultrasound. Based on the presence of pancytopenia, hyperferritinemia, and hemophagocytosis by a bone marrow examination, she was diagnosed with macrophage activation syndrome (MAS)/hemophagocytic lymphohistiocytosis (HLH) which was refractory to glucocorticoid pulse therapy. The combination of intravenous cyclosporine A with glucocorticoids was able to successfully control the disease activity of AOSD-related AAC and MAS/HLH.

Excessive postoperative scarring halts the effectiveness of glaucoma surgery and still remains a challenging problem. The purpose of this study was to develop a PLA-PEG-based drug delivery system with cyclosporine A or everolimus for wound healing modulation. Methods: PLA-PEG implants saturation with cyclosporine A or everolimus as well as their further in vitro release were analyzed. Anti-proliferative activity and cytotoxicity of the immunosuppressants were studied in vitro using human Tenon’s fibroblasts. Thirty-six rabbits underwent glaucoma filtration surgery with the application of sham implants or samples saturated with cyclosporine A or everolimus. The follow-up period was six months. A morphological study of the surgery area was also performed at seven days, one, and six months post-op. Results: PLA-PEG implants revealed a satisfactory ability to cumulate either cyclosporine A or everolimus. The most continuous period of cyclosporine A and everolimus desorption was 7 and 13 days, respectively. Immunosuppressants demonstrated marked anti-proliferative effect regarding human Tenon’s fibroblasts without signs of cytotoxicity at concentrations provided by the implants. Application of PLA-PEG implants saturated with immunosuppressants improved in vivo glaucoma surgery outcomes. Conclusions: Prolonged delivery of either cyclosporine A or everolimus by means of PLA-PEG implants represents a promising strategy of wound healing modulation in glaucoma filtration surgery.

Rationale Calcineurin is a protein regulating cytokine expression in T lymphocytes and calcineurin inhibitors such as cyclosporine A (CsA) are widely used for immunosuppressive therapy. It also plays a functional role in distinct neuronal processes in the central nervous system. Disturbed information processing as seen in neuropsychiatric disorders is reflected by deficient sensorimotor gating, assessed as prepulse inhibition (PPI) of the acoustic startle response (ASR). Objective Patients who require treatment with immunosuppressive drugs frequently display neuropsychiatric alterations during treatment with calcineurin inhibitors. Importantly, knockout of calcineurin in the forebrain of mice is associated with cognitive impairments and symptoms of schizophrenia-like psychosis as seen after treatment with stimulants. Methods The present study investigated in rats effects of systemic acute and subchronic administration of CsA on sensorimotor gating. Following a single injection with effective doses of CsA, adult healthy male Dark Agouti rats were tested for PPI. For subchronic treatment, rats were injected daily with the same doses of CsA for 1 week before PPI was assessed. Since calcineurin works as a modulator of the dopamine pathway, activity of the enzyme tyrosine hydroxylase was measured in the prefrontal cortex and striatum after accomplishment of the study. Results Acute and subchronic treatment with the calcineurin inhibitor CsA disrupted PPI at a dose of 20 mg/kg. Concomitantly, following acute CsA treatment, tyrosine hydroxylase activity was reduced in the prefrontal cortex, which suggests that dopamine synthesis was downregulated, potentially reflecting a stimulatory impact of CsA on this neurotransmitter system. Conclusions The results support experimental and clinical evidence linking impaired calcineurin signaling in the central nervous system to the pathophysiology of neuropsychiatric symptoms. Moreover, these findings suggest that therapy with calcineurin inhibitors may be a risk factor for developing neurobehavioral alterations as observed after the abuse of psychomotor stimulant drugs.

BACKGROUND Vernal keratoconjunctivitis (VKC) is a rare chronic conjunctivitis characterized by a predominantly eosinophil-mediated inflammatory disorder that could develop critical complications such as blindness. Oxidative stress plays a pivotal role in the pathogenesis of several allergic diseases. The role of oxidative stress has been hypothesized in VKC, but no study explored this issue. Furthermore, cyclosporine A (CsA) exerts an anti-inflammatory and antioxidant action on the conjunctiva. This study aimed to assess the oxidative stress in VKC patients and controls and to study the effect of CsA on oxidative stress in these subjects. METHODS Thirty-six consecutive children, including 12 VKC (nine males, 75%; mean age 10.17; SD ± 2.48) patients without treatment, 12 VKC patients treated with CsA (nine males, 75%; mean age 9.08; SD ± 2.75), and 12 controls (CT) (seven males, 58%; mean age 8.58; SD ± 1.78), were recruited. A cross-sectional study was performed to compare H2 O2 in the serum with that in the tears of these children. RESULTS Compared with CT and VKC children treated with CsA, VKC untreated children had significantly higher values of hydrogen peroxide (H2 O2 ) in the serum and the tears. No significant differences were observed between CT and VKC treated with CsA. A significant correlation was found at the linear regression analysis between serum and tear H2 O2 levels. CONCLUSION This study provides the first report attesting that patients with VKC have high oxidative stress; furthermore, it suggests that CsA could have an anti-inflammatory and antioxidant action that could be useful to prevent the poor VKC outcome.

A 40-year-old man had been treated for Behçet’s disease (BD) with cyclosporine A (CsA) for 14 years. He presented multiple lymphadenopathies with fever. Histological examination of surgical biopsy showed other iatrogenic immunodeficiency-associated lymphoproliferative disorders, diffuse large B-cell lymphoma type with positivity for Epstein-Barr virus encoding RNA-1 (EBER-1). BCL2-IgH, BCL6-IgH, and MYC-IgH translocations were not detected. CsA was stopped, and R-CHOP therapy was initiated. However, his lymphoma was chemotherapy resistant and rapidly progressed. To the best of our knowledge, this is the first case of diffuse large B-cell lymphoma that occurred in a BD patient treated with CsA reported in English. Both BD and CsA are associated with the pathogenesis of lymphoma. We also describe extremely rare cases in the form of a literature review.

Background/purpose: Oct4, a key transcription factor, could reprogram human somatic fibroblasts into embryonic stem cell-like pluripotent cells. The exact mechanism of cyclosporine A (CsA)-induced gingival overgrowth is still unclear. The aim of this study was to investigate the effects of CsA on the expression of Oct4 in cultured human gingival fibroblasts (HGFs) in vitro. Materials and methods: The effects of CsA on HGFs were used to elucidate whether Oct4 expression could be induced by CsA by using quantitative real-time reverse transcription-polymerase chain reaction and western blot. Cell growth in CsA-treated HGFs with Oct4 lentiviral-mediated shRNAi knockdown was evaluated by tetrazolium bromide reduction assay. Results: CsA was found to upregulate Oct4 transcript in a dose-dependent manner (p

Korean ginseng (Panax ginseng) is associated with a variety of therapeutic effects, including antioxidative, anti-inflammatory, vasorelaxative, antiallergic, antidiabetic, and anticancer effects. Accordingly, the use of ginseng has reached an all-time high among members of the general public. However, the safety and efficacy of ginseng in transplant recipients receiving immunosuppressant drugs have still not been elucidated. Transplantation is the most challenging and complex of surgical procedures and may require causation for the use of ginseng. In this regard, we have previously examined the safety, immunological benefits, and protective mechanisms of ginseng with respect to calcineurin inhibitor-based immunosuppression, which is the most widely used regimen in organ transplantation. Using an experimental model of calcineurin inhibitor-induced organ injury, we found that ginseng does not affect drug levels in the peripheral blood and tissue, favorably regulates immune response, and protects against calcineurin inhibitor-induced nephrotoxicity and pancreatic islet injury. On the basis of our experimental studies and a review of the related literature, we propose that ginseng may provide benefits in organ transplant recipients administered calcineurin inhibitors. Through the present review, we aimed to briefly discuss our current understanding of the therapeutic benefits of ginseng related to transplant patient survival. Keywords: cyclosporine A, calcineurin inhibitor, ginseng, transplantation, tacrolimus

Cyclosporine A (CsA) preconditioning is known to target mitochondrial permeability transition pore and protect renal function after ischemia reperfusion (IR). The upregulation of heat-shock protein 70 (Hsp70) expression after CsA injection is thought to be associated with renal protection. The aim of this study was to test the effect of Hsp70 expression on kidney and mitochondria functions after IR. Mice underwent a right unilateral nephrectomy and 30 min of left renal artery clamping, performed after CsA injection and/or administration of the Hsp70 inhibitor. Histological score, plasma creatinine, mitochondrial calcium retention capacity, and oxidative phosphorylation were assessed after 24 h of reperfusion. In parallel, we used a model of hypoxia reoxygenation on HK2 cells to modulate Hsp70 expression using an SiRNA or a plasmid. We assessed cell death after 18 h of hypoxia and 4 h of reoxygenation. CsA significantly improved renal function, histological score, and mitochondrial functions compared to the ischemic group but the inhibition of Hsp70 repealed the protection afforded by CsA injection. In vitro, Hsp70 inhibition by SiRNA increased cell death. Conversely, Hsp70 overexpression protected cells from the hypoxic condition, as well as the CsA injection. We did not find a synergic association between Hsp70 expression and CsA use. We demonstrated Hsp70 could modulate mitochondrial functions to protect kidneys from IR. This pathway may be targeted by drugs to provide new therapeutics to improve renal function after IR.

Immune thrombocytopenia (ITP) and autoimmune hemolytic anemia (AIHA) show good responses to frontline steroids. About two-third of cases relapse and require second-line treatment, including rituximab, mainly effective in AIHA, and thrombopoietin-receptor agonists (TPO-RAs) in ITP, while the use of splenectomy progressively decreased due to concerns for infectious/thrombotic complications. For those failing second line, immunosuppressants may be considered.The aim of this study was to evaluate the efficacy of cyclosporine treatment in patients with ITP and AIHA.In this retrospective study, we evaluated the efficacy and safety of cyclosporine A (CyA) in ITP (Responses were classified as partial [Hb 10 or at least 2 g/dl increase from baseline, platelets (PLT) 30 × 10The median time from diagnosis to CyA was 35 months (3-293), and patients had required a median of 4 (1-8) previous therapy lines. Median duration of CyA was 28 (2-140) months and responses were achieved in 86% of ITP and 50% of AIHA subjects. Responders could reduce or discontinue concomitant treatment and resolved PLT fluctuations on TPO-RA. CyA was generally well tolerated, and only two serious infectious complications in elderly patients on concomitant steroids suggesting caution in this patient population.CyA may be advisable in ITP, which is not well controlled under TPO-RA, and in AIHA failing rituximab, particularly if ineligible in clinical trial.

This study aimed to develop a cyclosporine A (CsA)-loaded ternary solid dispersion (tSD/CsA) to improve the storage stability of a solid dispersion (SD) system and the oral absorbability of CsA. Hydroxypropyl cellulose (HPC) and hydroxypropyl methylcellulose acetate succinate (HPMCAS) were selected as carrier materials of tSD, and tSD/CsA was prepared with a fine droplet drying process, a powderization technology that employs an inkjet head. The physicochemical properties of tSD/CsA were evaluated in terms of morphology, storage stability, dissolution behavior, and mucoadhesive property. After the oral administration of CsA samples (10 mg-CsA/kg) to rats, the plasma concentration of CsA was monitored to estimate oral absorbability. tSD/CsA comprised uniform shriveled particles with a diameter of 3.4 mm and span factor of 0.4, which is a parameter to estimate the particle size distribution. Although HPC-based binary SD showed marked aggregation of the particles after storage under 40 °C/75% relative humidity, there were no significant aggregations of tSD/CsA, due to the relatively low hygroscopic property of HPMCAS. The pH-dependent release of CsA with improved dissolution was observed in tSD/CsA. In the in vitro mucoadhesive evaluation using a mucin disk, tSD/CsA exhibited a better mucoadhesive property than HPC-based SD, possibly leading to prolonged retention of tSD particles in the gastrointestinal tract after oral administration. Orally-dosed tSD/CsA in rats resulted in significantly improved oral absorption of CsA, as evidenced by a 27-fold higher bioavailability than amorphous CsA. tSD/CsA may be a promising dosage option to improve the storage stability of a SD system and the biopharmaceutical properties of CsA.

Cyclosporine A (CsA) as an eye drop is an effective treatment for dry eye. However, it has potential side effects and a short ocular residence time. To overcome these obstacles, we developed a cellulose acetate phthalate-based pH-responsive contact lens (CL) loaded with CsA (CsA-CL). The CsA was continuously released from the CsA-CL at physiological conditions (37 °C, pH 7.4) without an initial burst. CsA was well-contained in the selected storage condition (4 °C, pH 5.4) for as long as 90 days. In safety assays, cytotoxicity, ocular irritation, visible light transmittance, and oxygen permeability were in a normal range. CsA concentrations in the conjunctiva, cornea, and lens increased over time until 12 h. When comparing the therapeutic efficacy between the normal control, experimental dry eye (EDE), and treatment groups (CsA eye drop, naïve CL, and CsA-CL groups), the tear volume, TBUT, corneal fluorescein staining at 7 and 14 days, conjunctival goblet cell density, and corneal apoptotic cell counts at 14 days improved in all treatment groups compared to EDE, with a significantly better result in the CsA-CL group compared with other groups (all p < 0.05). The CsA-CL could be an effective, stable, and safe option for inflammatory dry eye.

This study reports two therapeutic peptides, insulin (INS, as a hydrophilic model peptide) and cyclosporine A (CysA, as a hydrophobic one), that can be administrated through a transdermal or dermal route by using spicule-based topical delivery systems in vitro and in vivo. We obtained a series of spicules with different shapes and sizes from five kinds of marine sponges and found a good correlation between the skin permeability enhancement induced by these spicules and their aspect ratio L/D. In the case of INS, Sponge Haliclona sp. spicules (SHS) dramatically increased the transdermal flux of INS (457.0 ± 32.3 ng/cm2/h) compared to its passive penetration (5.0 ± 2.2 ng/cm2/h) in vitro. Further, SHS treatment slowly and gradually reduced blood glucose to 13.1 ± 6.3% of the initial level in 8 h, while subcutaneous injection resulted in a rapid blood glucose reduction to 15.9 ± 1.4% of the initial level in 4 h, followed by a rise back to 75.1 ± 24.0% of the initial level in 8 h. In the case of CysA, SHS in combination with ethosomes (SpEt) significantly (p < 0.05) increased the accumulation of CysA in viable epidermis compared to other groups. Further, SpEt reduced the epidermis thickness by 41.5 ± 9.4% in 7 days, which was significantly more effective than all other groups. Spicule-based topical delivery systems offer promising strategies for delivering therapeutic peptides via a transdermal or dermal route.

Vernal keratocongiuntivitis (VKC) is a chronic inflammatory disease affecting the ocular conjunctiva and cornea. It is a rare and underestimated pathology, whose missed or delayed diagnosis can lead to the development of serious ocular complications. Moreover, despite VKC symptoms are well known, they can overlap and be mistaken with allergic conjunctivitis. In fact, diagnostic criteria and severity grading are not standardized yet. The pathogenesis of VKC is still controversial and it is difficult to identify a single mechanism underlying the chronic ocular inflammation. Different studies hypothesized both allergies and autoimmune diseases and also oxidative stress contribute significantly to the origin of the disease. However, the unclear pathogenesis and the lack of specific disease biomarkers make treatment a challenge. The standard therapy includes antihistamines, anti-inflammatory and immunosuppressant drugs and novel therapies are currently under investigation. However, considering treatment guidelines and recommendations are not well defined yet, therapy should be personalized on the clinical features of the patient. This paper provides an overview of the VKC and updates on the challenges that need to be addressed in the future to improve the management of the patient with this disease and improve his quality of life.

Following organ transplantation, it is essential that immune tolerance is induced in the graft recipient to reduce the risk of rejection and avoid complications associated with the long-term use of immunosuppressive drugs. Immature dendritic cells (DCs) are considered to promote transplant tolerance and may minimize the risk of graft rejection. The aim of the study was to evaluate the effects of immunosuppressive agents: rapamycin (Rapa) and cyclosporine A (CsA) on generation of human tolerogenic DCs (tolDCs) and also to evaluate the ability of these cells to induce mechanisms of immune tolerance. tolDCs were generated in the environment of Rapa or CsA. Next, we evaluated the effects of these agents on surface phenotypes (CD11c, MHC II, CD40, CD80, CD83, CD86, CCR7, TLR2, TLR4), cytokine production (IL-4, IL-6, IL-10, IL-12p70, TGF-β), phagocytic capacity and resistant to lipopolysaccharide activation of these DCs. Moreover, we assessed ability of such tolDCs to induce T cell activation and apoptosis, Treg differentiation and production of Th1- and Th2-characteristic cytokine profile. Data obtained in this study demonstrate that rapamycin is effective at generating maturation-resistant tolDCs, however, does not change the ability of these cells to induce mechanisms of immune tolerance. In contrast, CsA affects the ability of these cells to induce mechanisms of immune tolerance, but is not efficient at generating maturation-resistant tolDCs. Supplementary Information The online version contains supplementary material available at 10.1007/s00005-021-00632-7.

Isolated human amniotic cells (hAC) could be used as a source of immunomodulatory factors in regenerative medicine and transplantation. However, in previous experimental studies, native hAC administered to skin graft recipients did not induce graft immunotolerance. To strengthen the immunomodulatory properties of hAC prior to administration to the recipient, we activated them ex vivo using pro-inflammatory cytokines. In this study, we compared the transplantation efficiency of skin allografts (mouse to mouse) and xnografts (rat to mouse) in recipient mice divided into three main groups receiving: 1. Placebo (control group); 2. Cyclosporine A (CsA) [10 or 50 mg/kg body weight (bw)]; 3. suspension of hAC activated ex vivo by IL-1β and INFγ, administered into a tail vein or subcutaneously. During 15 days of observation, hAC administered intravenously or subcutaneously after allotransplantation appeared to be as safe and efficient as CsA at the dose of 10 mg/kg bw in preventing rejection of skin allo- and xenografts. After xenotransplantation, however, only hAC administered intravenously prevented rejection to an extent comparable to CsA. Both CsA (10 mg/kg bw) and activated hAC reduced inflammatory infiltration in the skin (after intravenous injection) and did not increase the concentration of the inflammation marker SAP in serum or percentage of leukocytes in blood. Finally, we concluded that administration of activated hAC is safe and efficient in the presented animal model of skin allo- and xenotransplantation in a route-dependent manner. Activated hAC injected intravenously exhibit an immunosuppressive effect comparable to CsA administered at the dose of 10 mg/kg bw in both allo- and xenotransplantation.

In this thesis, we addressed the role of cellular metabolism in maturation of hiPSC-ECs. In Chapter 2, we compare hiPSC derived EC functionality and metabolism with primary human microvascular endothelial cells (hMVEC) determining the presence of a luminal glycocalyx as a main functional target. This chapter presents new insights in mitochondrial dysfunction of hiPSC-EC, limiting their ability to produce sufficient glycocalyx and align to shear stress. Underlying the mitochondrial dysfunction, we found that hiPSC-EC have an open mitochondrial permeability transition pore (mPTP), indicating mitochondrial immaturity. By closing the mPTP during differentiation with Cyclosporin-A (CsA), binding to cyclophilin D of the mPTP, we were able to mature mitochondria and improve functionality of these cells, resulting in a reduction in ROS, increased glycocalyx production and the therefore providing iPSC-ECs the ability to align to shear stress. Chapter 3 Continues with the comparison of hiPSC-EC with hMVEC, focusing on von Willebrand Factor (VWF) and the production of Weibel Palade Bodies (WPB). Testing several differentiation protocols and even after addition of CsA, hiPSC-EC were found to lack mature WPBs. We showed that neither shear stress nor co-culture with pericytes could induce WPB formation. By further studying the metabolism with NMR we found that hiPSC-EC have a reduced glycolysis and lactate production and an increased intracellular pH (pHi). This coincides with a reduced expression of the proton coupled monocarboxylate transporter MCT1, which transports H+ and lactate into the cell to keep pHi in balance. Reducing the intracellular pH led to increased presence of functional VWF and maturation of WPB in hiPSC-ECs. In Chapter 4 we addressed the question how hiPSC-EC maintain their redoxbalance and produce enough ATP, since the vast majority of ATP and antioxidants of EC are obtained by glycolysis, which is significantly reduced in iPSC-ECs. We found that hiPSC-EC rely mainly on free fatty acid oxidation and presumably use NADPH do maintain their redox balance. This alternative metabolic state in hiPSCEC was found to be independent of the high expression of the transcriptional coactivator peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α), which regulates fatty acid storage, glucose metabolism, lipid uptake and mitochondrial biogenesis. Since PGC1α is directly activated by ROS and is the master regulator of the energy metabolism, the high levels of PGC1α expression in hiPSCECcould also be a consequence instead of the cause of the observed differences in metabolism. In Chapter 5, we studied the immunogenic surface of hiPSC-ECs, since transplantation induced rejection by the host immune system is an essential hurdle in usage of IPSC derived tissue. Previous studies suggested that IPSC derived cell recognition by the host immune system is diminished compared to transplantation of allogenic human alternatives. On the other hand, endothelial surface MHC class 1 and 2 molecules do play an active, ‘APC like’, role in adult immunity. Therefore, we characterized hiPSC-EC surface immune complexes, unstimulated and after cytokine stimulation. In addition we tested how the observed difference in expression could influence CD8 T-cell activation. Furthermore, we characterized the expression of complement inhibitors on the cell surface, necessary for the protection against unprovoked complement activation in the blood. Chapter 6 provides a summary and discussion of the observations in this thesis, including future perspectives on studying metabolism and metabolepigenetics in iPSC derived kidney organoids by mass spectrometry imaging.

Cyclosporine A is very effective in treatment of many ophthalmic pathologies like immunological and inflammatory diseases of the cornea, conjunctive or uvea. It is also used to prevent corneal graft rejection and to treat dry eye syndrome. Local ocular administration significantly reduces the risk of systemic side effects. Currently on the market are two products in the form of eye drops: Restasis® (0.05% of cyclosporine) in US and Ikervis® (0.1% of cyclosporine) in the EU. Both are in the form of oil-in-water emulsion. Their availability is still limited and cyclosporine concentration is low. This is a reason that compounded formulations are prepared in hospital and retail pharmacies. The pharmacists do not have an access to the substance and use injectable (50 mg/ml cyclosporine), oral solutions (100 mg/ml cyclosporine) or even oral capsules (25-100 mg/capsule) as the source of cyclosporine. That allows to use of cyclosporine for topical ophthalmic applications in varies formulations, indications, concentration and posology. Due to the fact that cyclosporine is a highly lipophilic and poorly water soluble, it is mostly compounded and administered as an oily solution. Unfortunately, that dosage form is poorly tolerated after administration to the eye, mainly due to the fact of blurring vision commonly observed because of the high viscosity of the oils. However, for many patients use of compounded eye drops with cyclosporine can be the only hope to alleviate the symptoms of the disease, and sometimes even to avoid vision loss. When pharmaceutical compounding of eye drops with cyclosporine, the problem is the presence of auxiliary substances such as, for example, Cremophor, ethanol evaporation, or obtaining the exact dose of the active substance from capsules. This article gives an overview of different approaches for compounding of the cyclosporine eye drops in hospital and retail pharmacies using marketed products off-label. Benefits and the limitations of each method are presented with comments regarding the proper drug compounding.

Regulatory T cells (Tregs) play a crucial role in the control of the initiation and progression of type 1 diabetes (T1D). Various immunological interventions including those to ex vivo expansion Tregs transfer, in vivo induction of peripherally derived Treg (pTreg) have been considered as promising approaches for T1D therapy. In this study, we developed a novel tolerogenic vaccine using four autoantigenic peptides of islet-derived with cyclosporine A (CsA) as the pTreg inducer, designated as GAD-IN+CsA. This vaccine immunized into prediabetic NOD mice subcutaneously could induce IL-10 and TGF-β expressing pTregs and lead to suppressing autoreactive T cells responses, resulting in the prevention of T1D in these animals. Furthermore, we demonstrated that CsA with autoantigenic peptides modulates dendritic cells (DCs) to become immature IL-10hiCD40lo DCs. Such modulated DCs could foster naïve CD4+CD25− T cell into Tregs when presenting antigen peptides in vitro. This novel approach offers an alternative strategy to induce pTregs to treat T1D.

Cyclosporin A (CsA) is a common immunosuppressant, but its use is limited as it can cause chronic kidney injury. Oxidative stress and apoptosis play a key role in CsA‐induced nephrotoxicity. This study investigated the protective effect of 5‐aminolevulinic acid and iron (5‐ALA/SFC) on CsA‐induced injury in murine proximal tubular epithelial cells (mProx24). 5‐ALA/SFC significantly inhibited apoptosis in CsA‐treated mProx24 cells with increases in heme oxygenase (HO)‐1, nuclear factor E2‐related factor 2 (Nrf2), and p38, and Erk‐1/2 phosphorylation. Moreover, 5‐ALA/SFC suppressed production of reactive oxygen species in CsA‐exposed cells and inhibition of HO‐1 suppressed the protective effects of 5‐ALA/SFC. In summary, 5‐ALA/SFC may have potential for development into a treatment for the anti‐nephrotoxic/apoptotic effects of CsA.

Calcineurin inhibitors (CNI) are the pillars of immunosuppression in transplantation. However, they display a potent nephrotoxicity whose mechanisms remained widely unsolved. We used an untargeted quantitative proteomic approach (iTRAQ technology) to highlight new targets of CNI in renal proximal tubular cells (RPTCs). CNI‐treated RPTCs proteome displayed an over‐representation of actin‐binding proteins with a CNI‐specific expression profile. Cyclosporine A (CsA) induced F‐actin remodeling and depolymerization, decreased F‐actin‐stabilizing, polymerization‐promoting cofilin (CFL) oligomers, and inhibited the G‐actin‐regulated serum response factor (SRF) pathway. Inhibition of CFL canonical phosphorylation pathway reproduced CsA effects; however, S3‐R, an analogue of the phosphorylation site of CFL prevented the effects of CsA which suggests that CsA acted independently from the canonical CFL regulation. CFL is known to be regulated by the Na+/K+‐ATPase. Molecular docking calculations identified two inhibiting sites of CsA on Na+/K+‐ATPase and a 23% decrease in Na+/K+‐ATPase activity of RPTCs was observed with CsA. Ouabain, a specific inhibitor of Na+/K+‐ATPase also reproduced CsA effects on actin organization and SRF activity. Altogether, these results described a new original pathway explaining CsA nephrotoxicity.

Cyclosporine A (CsA) is a nephrotoxicant that causes fibrosis via induction of epithelial–mesenchymal transition (EMT). The flavonoid chrysin has been reported to have anti-fibrotic activity and inhibit signaling pathways that are activated during EMT. This study investigated the nephroprotective role of chrysin in the prevention of CsA-induced renal fibrosis and elucidated a mechanism of inhibition against CsA-induced EMT in proximal tubule cells. Treatment with chrysin prevented CsA-induced renal dysfunction in Sprague Dawley rats measured by blood urea nitrogen (BUN), serum creatinine and creatinine clearance. Chrysin inhibited CsA-induced tubulointerstitial fibrosis, characterized by reduced tubular damage and collagen deposition. In vitro, chrysin significantly inhibited EMT in LLC-PK1 cells, evidenced by inhibition of cell migration, decreased collagen expression, reduced presence of mesenchymal markers and elevated epithelial junction proteins. Furthermore, chrysin co-treatment diminished CsA-induced TGF-β1 signaling pathways, decreasing Smad 3 phosphorylation which lead to a subsequent reduction in Snail expression. Chrysin also inhibited activation of the Akt/ GSK-3β pathway. Inhibition of both pathways diminished the cytosolic accumulation of β-catenin, a known trigger for EMT. In conclusion, flavonoids such as chrysin offer protection against CsA-induced renal dysfunction and interstitial fibrosis. Chrysin was shown to inhibit CsA-induced TGF-β1-dependent EMT in proximal tubule cells by modulation of Smad-dependent and independent signaling pathways.

Immunosuppressants are a mandatory therapy for transplant patients to avoid rejection of the transplanted organ by the immune system. However, there are several known side effects, including alterations of the vasculature, which involve a higher occurrence of cardiovascular events. While the effects of the commonly applied immunosuppressive drugs cyclosporine A (CsA) and tacrolimus (Tac) on mature endothelial cells have been addressed in several studies, we focused our research on the unexplored effects of CsA and Tac on endothelial colony-forming cells (ECFCs), a subgroup of endothelial progenitor cells, which play an important role in vascular repair and angiogenesis. We hypothesized that CsA and Tac induce functional defects and activate an inflammatory cascade via NF-κB signaling in ECFCs. ECFCs were incubated with different doses (0.01 µM–10 µM) of CsA or Tac. ECFC function was determined using in vitro models. The expression of inflammatory cytokines and adhesion molecules was explored by quantitative real-time PCR and flow cytometry. NF-κB subunit modification was assessed by immunoblot and immunofluorescence. CsA and Tac significantly impaired ECFC function, including proliferation, migration, and tube formation. TNF-α, IL-6, VCAM, and ICAM mRNA expression, as well as PECAM and VCAM surface expression, were enhanced. Furthermore, CsA and Tac led to NF-κB p65 subunit phosphorylation and nuclear translocation. Pharmacological inhibition of NF-κB by parthenolide diminished CsA- and Tac-mediated proinflammatory effects. The data of functional impairment and activation of inflammatory signals provide new insight into mechanisms associated with CsA and Tac and cardiovascular risk in transplant patients.

Due to the widely application of Cyclosporine A (CsA) as an immunosuppressant in clinic, it is necessary to study its potential toxicity. Therefore, we used zebrafish as a model animal to evaluate the toxicity of CsA on embryonic development. Exposure of zebrafish embryos to CsA at concentrations of 5 mg/L, 10 mg/L, and 15 mg/L from 12 hpf to 72 hpf resulted in abnormal embryonic development, including cardiac malformation, pericardial edema, decreased heart rate, decreased blood flow velocity, deposition at yolk sac, shortened body length, and increased distance between venous sinus and arterial bulb (SV-BA). The expression of genes related to cardiac development was disordered, and the apoptotic genes were up-regulated. Oxidative stress level was up-regulated and accumulated in pericardium in a dose-dependent manner. Astaxanthin (ATX) treatment could significantly alleviate zebrafish heart defects. CsA induced up-regulation of Wnt signaling in zebrafish, and IWR-1, an inhibitor of Wnt signaling pathway, could effectively rescue the heart defects induced by CsA. Together, our study indicated that CsA induced cardiac developmental toxicity in zebrafish larvae through up-regulating oxidative stress and Wnt signaling, contributing to a more comprehensive evaluation of the safety of the drug.

The use of cyclosporine A (CsA) in transplant recipients is limited due to its side effects of causing severe hypertension. We have previously shown that CsA increases the activity of the epithelial sodium channel (ENaC) in cultured distal nephron cells. However, it remains unknown whether ENaC mediates CsA-induced hypertension and how we could prevent hypertension. Our data show that the open probability of ENaC in principal cells of split-open cortical collecting ducts was significantly increased after treatment of rats with CsA; the increase was attenuated by lovastatin. Moreover, CsA also elevated the levels of intracellular cholesterol (Cho), intracellular reactive oxygen species (ROS) via activation of NADPH oxidase p47phox, serum- and glucocorticoid-induced kinase isoform 1 (Sgk1), and phosphorylated neural precursor cell–expressed developmentally downregulated protein 4–2 (p-Nedd4-2) in the kidney cortex. Lovastatin also abolished CsA-induced elevation of α-, ß-, and γ-ENaC expressions. CsA elevated systolic blood pressure in rats; the elevation was completely reversed by lovastatin (an inhibitor of cholesterol synthesis), NaHS (a donor of H2S which ameliorated CsA-induced elevation of reactive oxygen species), or amiloride (a potent ENaC blocker). These results suggest that CsA elevates blood pressure by increasing ENaC activity via a signaling cascade associated with elevation of intracellular ROS, activation of Sgk1, and inactivation of Nedd4-2 in an intracellular cholesterol-dependent manner. Our data also show that NaHS ameliorates CsA-induced hypertension by inhibition of oxidative stress.

Background: Cyclosporine A (CsA) is a potent immunosuppressant widely used to prevent renal post transplantation rejection. Gingival overgrowth (GO) is among various side effects of the long-term administration of CsA. Up to 90% of the patients under CsA therapy has been reported to develop CsA-induced GO. Objectives: The aim of the present prospective pilot study is to determine the local and systemic effects of Cyclosporine A (CsA) on the severity of gingival overgrowth and its relationship with periodontal parameters in post-transplant renal patients Methods: Twenty post-transplant renal patients, 12 females and 8 males, presenting gingival overgrowth were selected from Rizk Hospital’s clinic in Beirut. Patient’s CsA plaque levels were evaluated when CsA is administered by syrup and capsules mode. Periodontal parameters including gingival overgrowth, papillary bleeding, plaque and gingival indices were assessed for all patients. Results: Plaque concentration CsA levels, when administered in syrup mode, affected significantly the severity of gingival overgrowth as opposed to the administration by capsule mode. Significant correlations between severity of gingival overgrowth on one hand and plaque index, gingival index, and papillary bleeding index on the other hand were only observed in the Capsule group but not in the syrup group. A significant relationship was established between the severity of gingival overgrowth and all periodontal parameters (gingival, papillary and plaque). Conclusion: The present study underlines CsA dental plaque local effect, as a co-factor, in the development of gingival overgrowth. Cyclosporine plaque accumulation acts as a reservoir in the gingival inflammation and the periodontal indices seem to be the most accurate parameters associated with gingival overgrowth severity. Plaque CsA concentrations could be considered as a risk factor for inflammation and gingival overgrowth depending on CsA delivery mode in renal transplant patients.

Acute lung injury (ALI), which is induced by renal ischemia-reperfusion (IR), is one of the leading causes of acute renal IR-related death. Obesity raises the frequency and severity of acute kidney injury (AKI) and ALI. Tanshinone IIA (TIIA) combined with cyclosporine A (CsA) was employed to lessen the lung apoptosis led by renal IR and to evaluate whether TIIA combined with CsA could alleviate lung apoptosis by regulating mitochondrial function through the PI3K/Akt/Bad pathway in obese rats. Hematoxylin-eosin (HE) staining was used to assess the histology of the lung injury. Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) was used to assess apoptosis of the lung. Electron microscopy was used to assess mitochondrial morphology in lung cells. Arterial blood gas and pulmonary function were used to assess the external respiratory function. Mitochondrial function was used to assess the internal respiratory function and mitochondrial dynamics and biogenesis. Western blot (WB) was used to examine the PI3K/Akt/Bad pathway-related proteins. TIIA combined with CsA can alleviate lung apoptosis by regulating mitochondrial function through the PI3K/Akt/Bad pathway in obese rats.

Immunosuppressive ability in human MSC donors has been shown to be variable and may be a limiting factor in MSC therapeutic efficacy in vivo. The importance of cytokine activation of mesenchymal stromal cells (MSCs) to facilitate their immunosuppressive function is well established. This study sought to further understand the interactions between MSCs and the commonly used calcineurin inhibitor cyclosporine A (CsA). The existing literature regarding approaches that use MSCs and cyclosporine are conflicting regarding the effect of CsA on MSC potency and function. Here, we clearly demonstrate that when added at the same time as MSCs, CsA negatively affects MSC suppression of T cell proliferation. However, licencing MSCs with IFNγ before addition of CsA protects MSCs from this negative effect. Notably, adding CsA to MSCs after IFNγ pre-stimulation enhances MSC production of IDO. Mechanistically, we identified that CsA reduces SOCS1 expression to facilitate enhanced IDO production in IFNγ pre-stimulated MSCs. Importantly, CsA exposure to IFNγ pre-stimulated MSC before administration, significantly enhanced the potency of MSCs in a human relevant humanised mouse model of acute Graft versus Host Disease. In summary, this study identified a novel licencing strategy to enhance MSC potency in vitro and in vivo. Supplementary Information The online version contains supplementary material available at 10.1186/s13287-021-02309-6.

Background Cystic echinococcosis (CE) is a disease caused by the larval stage of Echinococcus granulosus sensu lato (s.l.). The treatment of CE mainly relies on the use of benzimidazoles, which can commonly cause adverse side effects. Therefore, more efficient treatment options are needed. Drug repurposing is a useful approach for advancing drug development. We have evaluated the in vitro protoscolicidal effects of tropisetron and granisetron in E. granulosus sensu stricto (s.s.) and assessed the expression of the calcineurin (CaN) and calmodulin (CaM) genes, both of which have been linked to cellular signaling activities and thus are potentially promising targets for the development of drugs. Methods Protoscoleces (PSC) of E. granulosus (s.s.) (genotype G1) obtained from sheep hepatic hydatid cysts were exposed to tropisetron and granisetron at concentrations of 50, 150 and 250 µM for various periods of time up to 10 days. Cyclosporine A (CsA) and albendazole sulfoxide were used for comparison. Changes in the morphology of PSC were investigated by light microscopy and scanning electron microscopy. Gene expression was assessed using real-time PCR at the mRNA level for E. granulosus calcineurin subunit A (Eg-CaN-A), calcineurin subunit B (Eg-CaN-B) and calmodulin (Eg-CaM) after a 24-h exposure at 50 and 250 µM, respectively. Results At 150 and 250 µM, tropisetron had the highest protoscolicidal effect, whereas CsA was most effective at 50 µM. Granisetron, however, was less effective than tropisetron at all three concentrations. Examination of morphological alterations revealed that the rate at which PSC were killed increased with increasing rate of PSC evagination, as observed in PSC exposed to tropisetron. Gene expression analysis revealed that tropisetron at 50 μM significantly upregulated Eg-CaN-B and Eg-CaM expression while at 250 μM it significantly downregulated both Eg-CaN-B and Eg-CaM expressions; in comparison, granisetron decreased the expression of all three genes at both concentrations. Conclusions Tropisetron exhibited a higher efficacy than granisetron against E. granulosus (s.s.) PSC, which is probably due to the different mechanisms of action of the two drugs. The concentration-dependent effect of tropisetron on calcineurin gene expression might reflect its dual functions, which should stimulate future research into its mechanism of action and evaluation of its potential therapeutical effect in the treatment of CE. Graphical Abstract

The clinical application of cyclosporine A (CsA) as an immunosuppressive agent is limited by its organ toxicity. We aimed to evaluate the effectiveness of α-lipoic acid against CsA-induced hepatotoxicity and to delineate the underlying molecular mechanisms. Male Wistar rats (n = 24, 8 per each group) received the vehicle, CsA (25 mg/kg) and/or ALA (100 mg/kg, p.o.) for 3 weeks. Biochemical markers of liver function (serum ALT, AST, ALP < GGT), oxidative stress (MDA, TAC, SOD, GSH, Nrf2/HO-1), inflammation (NF-κB, CD68, iNOS, NO, COX-2), and apoptosis (caspase-3) were assessed in serum and tissue. Liver histological analysis using H&E and Sirius red was performed. The development of liver injury in CsA-treated animals was indicated by elevated levels of liver enzymes, oxidants/antioxidants imbalance, inflammatory cells infiltration, up-regulated expression of inflammatory mediators, and apoptosis. These changes were associated with altered architecture of hepatic cells and fibrous connective tissue. ALA co-administration protected against CsA-induced liver damage and ameliorated biochemical changes and cellular injury. In conclusion, ALA demonstrated hepatoprotective potential against CsA-induced liver injury through combating oxidative stress, inflammation, and apoptosis, highlighting ALA as a valuable adjunct to CsA therapy.

The immunosuppressant cyclosporine A (CSA) has been linked to serious renal toxic effects. Although 2-methoxyestradiol (2ME) possesses a wide range of pharmacological abilities, it suffers poor bioavailability after oral administration. The purpose of this study was to evaluate the potential of 2ME loaded D-ɑ-tocopheryl polyethylene glycol succinate (TPGS) micelles to prevent CSA-induced nephrotoxicity in rats. A 2ME-TPGS was prepared and showed particle size of 44.3 ± 3.5 nm with good entrapment efficiency and spherical structures. Male Wistar rats were divided into 5 groups, namely: Control, Vehicle, CSA, CSA + 2ME-Raw, and CSA + 2ME-Nano. CSA was injected daily at a SC dose of 20 mg/kg. Both 2ME-Raw and 2ME-Nano were given daily at oral doses of 5 mg/kg. Treatments continued for three successive weeks. 2ME-TPGS exerted significant protective effects against CSA nephrotoxicity. This was evidenced in ameliorating deterioration of renal functions, attenuation of pathological changes in kidney tissues, exerting significant anti-fibrotic, antioxidant, and anti-inflammatory effects together with significant anti-apoptotic effects. Western blot analyses showed both 2ME-Raw and 2ME-Nano significantly inhibited protein expression of TGF-β1 and phospho-ERK (p-ERK). It was observed that 2ME-TPGS, in almost all experiments, exerted superior protective effects as compared with 2ME-Raw. In conclusion, 2ME loaded in a TPGS nanocarrier possesses significant protective activities against CSA-induced kidney injury in rats. This is attributable to 2ME anti-fibrotic, antioxidant, anti-inflammatory, and anti-apoptotic activities which are mediated at least partly by inhibition of TGF-β1/p-ERK axis.

Renal ischemia–reperfusion (IR) injury and cyclosporine A (CsA) nephrotoxicity affect allograft function and survival. The prolonged effects and underlying mechanisms of erythropoietin derived cyclic helix B peptide (CHBP) and/or caspase-3 small interfering RNA (CASP-3siRNA) were investigated in mouse kidneys, as well as kidney epithelial cells (TCMK-1), subjected to transplant-related injuries. Bilateral renal pedicles were clamped for 30 min followed by reperfusion for 2 and 8 weeks, with/without 35 mg/kg CsA gavage daily and/or 24 nmol/kg CHBP intraperitoneal injection every 3 days. The ratio of urinary albumin to creatinine was raised by IR injury, further increased by CsA and lowered by CHBP at 2, 4, 6 and 8 weeks, whereas the level of SCr was not significantly affected. Similar change trends were revealed in tubulointerstitial damage and fibrosis, HMGB1 and active CASP-3 protein. Increased apoptotic cells in IR kidneys were decreased by CsA and CHBP at 2 and/or 8 weeks. p70 S6 kinase and mTOR were reduced by CsA with/without CHBP at 2 weeks, so were S6 ribosomal protein and GSK-3β at 8 weeks, with reduced CASP-3 at both time points. CASP-3 was further decreased by CHBP in IR or IR + CsA kidneys at 2 or 8 weeks. Furthermore, in TCMK-1 cells CsA induced apoptosis was decreased by CHBP and/or CASP-3siRNA treatment. Taken together, CHBP predominantly protects kidneys against IR injury at 2 weeks and/or CsA nephrotoxicity at 8 weeks, with different underlying mechanisms. Urinary albumin/creatinine is a good biomarker in monitoring the progression of transplant-related injuries. CsA divergently affects apoptosis in kidneys and cultured kidney epithelial cells, in which CHBP and/or CASP-3siRNA reduces inflammation and apoptosis.

International audience; The blood-brain barrier (BBB) is a highly restrictive barrier that preserves central nervous system homeostasis and ensures optimal brain functioning. Using BBB cell assays makes it possible to investigate whether a compound is likely to compromise BBBs functionality, thereby probably resulting in neurotoxicity. Recently, several protocols to obtain human brain-like endothelial cells (BLECs) from induced pluripotent stem cells (iPSCs) have been reported. Within the framework of the European MSCA-ITN in3 project, we explored the possibility to use an iPSC-derived BBB model to assess the effects of repeated dose treatment with chemicals, using Cyclosporine A (CsA) as a model compound. The BLECs were found to exhibit important BBB characteristics up to 15 days after the end of the differentiation and could be used to assess the effects of repeated dose treatment. Although BLECs were still undergoing transcriptional changes over time, a targeted transcriptome analysis (TempO-Seq) indicated a time and concentration dependent activation of ATF4, XBP1, Nrf2 and p53 stress response pathways under CsA treatment. Taken together, these results demonstrate that this iPSC-derived BBB model and iPSC-derived models in general hold great potential to study the effects of repeated dose exposure with chemicals, allowing personalized and patient-specific studies in the future.

Introduction: Patients with anti-melanoma differentiation-associated gene 5 (MDA5) antibody-positive amyopathic dermatomyositis (ADM) often develop rapidly progressive interstitial lung diseases (RP-ILD), with poor treatment success. Many studies have shown that this is the main cause of death in patients with anti-MDA5 antibody-positive ADM.Case Presentation: A 37-years-old woman developed a cough, shortness of breath, and a rash on both hands, which resembled Gottron's signs. Upon laboratory examination, the results were as follows: antinuclear antibody (ANA) positive; anti-Ro52 antibody positive; and anti-MDA5 antibody positive. Pulmonary high-resolution CT (HRCT) scan showed pulmonary interstitial inflammatory changes, and mediastinal and subcutaneous emphysema. She was finally diagnosed with anti-MDA5 antibody-positive ADM accompanied by RP-ILD. She was first given high-dose-steroid pulse therapy with methylprednisolone (500 mg per day for 3 days) followed by methylprednisolone (40 mg, daily), cyclosporine A (100 mg, twice per day), and hydroxychloroquine (200 mg, twice per day). Since her discharge from our hospital in March of 2018, she has maintained the methylprednisolone therapy (tapered to 10 mg daily), cyclosporine A (100 mg, twice per day), and hydroxychloroquine (200 mg, twice per day).Outcomes: Pulmonary HRCT scans taken on 4, 9, and 26 months after her discharge from our hospital showed that the interstitial pneumonitis had significantly improved and that mediastinal and subcutaneous emphysema had been gradually absorbed. The patient can now participate in regular work and activities of daily living.Conclusion: The treatment of methylprednisolone pulse therapy combined with cyclosporine A and hydroxychloroquine may be an option for the RP-ILD accompanied by anti-MDA-positive ADM. After the acute phase, this combination therapy strategy is helpful to the disease control of patients.

目的 研究非重型再生障碍性贫血（NSAA）患者接受环孢素A（CsA）治疗前后生活质量的变化，并分析生活质量改善的影响因素。 方法 收集2014年1月至2016年1月在北京协和医院门诊初次治疗、仅使用CsA的NSAA患者，在治疗前和治疗2年后分别填写SF-36量表，与正常中国人模型（常模）比较；并调查评估患者的年龄、性别、文化程度、家庭年收入、付费方式、依从性、治疗前一般健康状况及治疗2年后的疗效，分析其与生活质量改善的关系。 结果 共52例患者符合入组条件，其中男性27例（51.9％），女性25例（48.1％），中位年龄48（21～85）岁。CsA治疗2年，完全治疗反应（CR）15例（28.8％），部分治疗反应（PR）25例（48.1％），无治疗反应（NR）12例（23.1％），治疗总有效率（CR+PR）76.9％。患者治疗前SF-36量表躯体和心理健康得分差于常模（P值均0.05），精力（VT）、精神健康（MH）得分高于常模［VT：（58.8±20.1）分对（52.3±20.9）分，P＝0.023；MH：（65.9±17.6）分对（59.7±22.9）分，P＝0.014］。不同社会背景与生活质量改善均无显著相关性。起病时体能状况评分（ECOG评分）较低者生活质量改善更大。CsA治疗有效者生活质量均得到显著改善。 结论 NSAA患者生活质量低于常人，CsA治疗可以改善患者的生活质量，尤其是心理健康情况。不同社会背景的患者均可从治疗中获益，而起病较重者获益更大。

Background COVID‐19 pandemic causes high global morbidity and mortality, and better medical treatments to reduce mortality are needed. Objective To determine the added benefit of Cyclosporine‐A (CsA), to low‐dose steroid treatment, in patients with COVID‐19. Methods Open‐label, non‐randomized pilot study of patients with confirmed infection of SARS‐CoV2, hospitalized from April to May 2020 at a single center in Puebla, Mexico. Comparative treatment with steroids or CsA plus steroids. Pneumonia severity was assessed by clinical, laboratory, and lung tomography. The death rate was evaluated at 28 days. Results 209 adult patients were studied, 105 received CsA plus steroids (age 55.3 ±13.3; 69% men), and 104 steroids alone (age 54.06 ±13.8; 61% men). All patients received clarithromycin, enoxaparin, and methylprednisolone or prednisone up to 10 days. Patient´s death was associated with hypertension (RR=3.5) and diabetes (RR=2.3). Mortality was 22 and 35% for CsA and control groups (p=0.02), respectively, for all patients, and 24 and 48.5% for patients with moderate to severe disease (p=0.001). Higher cumulative clinical improvement was seen for the CsA group (Nelson Aalen curve, p=0.001, log‐rank test) in moderate to severe patients. The Cox proportional hazard analysis showed the highest HR improvement value of 2.15 (1.39‐3.34, 95%CI, p=0.0005) for CsA treatment in moderate to severe patients, and HR = 1.95 (1.35‐2.83, 95%CI, p=0.0003) for all patients. Conclusion CsA used as an adjuvant to steroid treatment for COVID‐19 patients showed to improve outcomes and reduce mortality, mainly in those with moderate to severe disease. Further investigation through controlled clinical trials is warranted.

Dry eye disease (DED) is a multifactorial disease of the ocular surface and tear film that has gained awareness as a public health problem. Characteristics of DED include tear film instability, hyperosmolarity, and ocular surface inflammation, which can occur independently or may be a sequela of numerous ocular diseases, ocular surgery or contact lens wear. Much has been learned about the impact of the disease to help affected individuals who report symptoms of poor vision, pain, and tearing. Recently, new research highlights the importance of the role of ocular surface inflammation and damage in DED—leading to a vicious cycle of inflammation as well as loss of tear film homeostasis. DED immunopathophysiology is characterized by four stages: initiation, amplification, recruitment, and re-initiation. Cyclosporine is proven to be a valuable ophthalmic therapeutic for DED through its immunomodulatory actions and regulation of the adaptive immune response. Cyclosporine mechanism of action is well described in the published literature and the myriad of benefits in all four stages lend a broad-based immunomodulatory function particularly suitable for addressing DED. Furthermore, cyclosporine has unique goblet cell density improvement capabilities as well as anti-apoptotic properties. Topical formulations of cyclosporine are centered around addressing the highly lipophilic nature of the molecule. The poor aqueous solubility of cyclosporine traditionally presented technical challenges in drug delivery to the ocular surface. Newer formulations such as cationic emulsions and nanomicellar aqueous solutions address formulation, tissue concentration, and drug delivery challenges.

Akhtar Rasul,1,* Muhammad Imran Khan,2,* Mujeeb Ur Rehman,1,* Ghulam Abbas,1,* Nosheen Aslam,3,* Shabbir Ahmad,4,* Khizar Abbas,5,* Pervaiz Akhtar Shah,6,* Muhammad Iqbal,7,* Ali Mohammad Ahmed Al Subari,6,* Talal Shaheer,8,* Shahid Shah9,* 1Department of Pharmaceutics, Faculty of Pharmaceutical Sciences, Government College University, Faisalabad, Pakistan; 2Riphah Institute of Pharmaceutical Sciences, Riphah International University, Lahore Campus, Lahore, Pakistan; 3Department of Biochemistry, Government College University, Faisalabad, Pakistan; 4Cancer Center, Faculty of Health Sciences, University of Macau, Taipa, Macau, People’s Republic of China; 5Department of Pharmacognosy, Faculty of Pharmacy, Bahauddin Zakariya University, Multan, Pakistan; 6University College of Pharmacy, University of the Punjab, Lahore, Pakistan; 7Faculty of Pharmacy, Gomal University, Dera Ismail Khan, Pakistan; 8Department of Pharmacognosy, Xian Jiaotong University, Xian, People’s Republic of China; 9Department of Pharmacy Practice, Faculty of Pharmaceutical Sciences, Government College University, Faisalabad, Pakistan*All authors contributed equally to this workCorrespondence: Akhtar Rasul Email akhtar.rasul@gcuf.edu.pkBackground: Cyclosporine A (CsA) is an exceptional immunosuppressant used for the treatment of immune disorders. Niosomal vesicles are promising drug carriers that are formed by self-association of nonionic surfactants and cholesterol in an aqueous phase. The objective of the study was to formulate combined nonionic surfactant based vesicles and to evaluate their in vitro characterization, release studies and in vivo studies.Materials and Methods: Five niosomal formulations (F7 to F11) were prepared using the thin film hydration method. The molar ratio of cholesterol and non-ionic surfactant taken was 1:1. In formulation F10, the combination of surfactants Span 20 and Brij 35 was used. The niosomes were characterized by zeta sizer and SEM for particle size analysis, in vitro drug release and stability studies. The pharmacokinetic studies were conducted on healthy albino rabbits.Results: The size of niosome was found in the range of 427.1 nm to 972.3 nm. SEM image of optimized formulations F10 exhibit the spherical nature of niosomal vesicles. DSC thermograms of niosomal formulations exhibited a broadened endothermic peak. The stability study exhibited that all formulations are stable and negligible change of vesicle size and entrapment was observed with time. The percentage drug release was significantly higher as compared to CsA plain dispersion for all niosomal formulations at pH 1.2 and 7.4. The release kinetic behavior showed that all preparations were best described by zero order and can release active ingredient in a sustained manner. The pharmacokinetic data showed the test formulation (F10) possessed greater bioavailability as compared to the reference formulation (CsA aqueous dispersion).Conclusion: The formulation F10 demonstrated a comparatively more delayed rate of release with enhanced dissolution as compared to a single surfactant scheme. The F10 formulation can be a remarkable nanotechnology for prolonged delivery of CsA orally with improved dissolution profile and bioavailability.Keywords: in vitro study, cyclosporine A, niosomes, nonionic surfactants

Cyclophilin D (CypD) is a mitochondrial peptidyl-prolyl cis-trans isomerase, well known for regulating the mitochondrial permeability transition pore (PTP), a non-specific large conductance pore whose opening leads to cell death and has been implicated in ischemia/reperfusion injury in multiple organs, in neurodegenerative disorders and in muscular dystrophies. While the main target of CypD is a matter of ongoing research, inhibiting CypD protects in models of those diseases making it an interesting therapeutic target. The present review focuses on post-translational modifications of CypD that have been identified by recent studies, which can alter the regulation of the PTP, and contribute to understanding the mechanisms of action of CypD.

The outbreak of pneumonia caused by a new coronavirus (SARS-CoV-2) occurred in December 2019, and spread rapidly throughout the world. There have been other severe coronavirus outbreaks worldwide, namely, severe acute respiratory syndrome (SARS-CoV) and Middle East respiratory syndrome (MERS-CoV). Because the genetic diversity of coronaviruses renders the design of vaccines complicated, broad spectrum-anti-coronavirus drugs have become a critical approach to control the coronavirus epidemic. Cyclophilin A is an important protein needed for coronavirus replication, and its inhibitor cyclosporine A has the ability to suppress coronavirus on a broad spectrum. CD147-S protein was found to be one route by which SARS-CoV-2 invades host cells, while CD147 was found to play a functional role in facilitating the infection of host cells by SARS-CoV. The CyPA/CD147 interaction may play a critical role in the ability of the SARS-CoV-2 virus to enter the host cells. However, cyclosporine A has immunosuppressive effects, so the conditions for its use as an antiviral drug are limited. As a result, cyclosporine A analogues without immunosuppressive side effects have attracted lots of interest. This review primarily discusses the drug development prospects of cyclophilin A as a therapeutic target for the treatment of coronavirus infection, especially coronavirus disease 2019 (COVID-19), and non-immunosuppressive cyclosporine analogues.

The cytochrome P450 enzyme CYP-sb21 from the rare actinomycete Sebekia benihana is capable of hydroxylating the immunosuppressive drug molecule cyclosporine A (CsA) primarily at the 4th N-methyl leucine (MeLeu4), giving rise to γ-hydroxy-N-methyl- l -Leu4-CsA (CsA-4-OH). This oxidative modification of CsA leads to dramatically reduced immunosuppressive activity while retaining the hair growth-promoting side-effect, thus demonstrating great application potential in both pharmaceutical and cosmetic industries. However, this P450 enzyme also hydroxylates CsA at the unwanted position of the 9th N-methyl leucine (MeLeu9), indicating that the regioselectivity needs to be improved for the development of CsA-4-OH into a commercial hair growth stimulator. Herein, we report the crystal structure of CYP-sb21 in its substrate-free form at 1.85 A. Together with sequence and 3D structure comparisons, Autodock-based substrate docking, molecular dynamics (MD) simulation, and site-directed mutagenesis, we identified a number of key residues including R294, E264, and M179 that can improve catalytic efficiency or change the regioselectivity of CYP-sb21 towards CsA, setting the stage for better enzymatic preparation of CsA-4-OH. This study also provides new insights into the substrate recognition and binding mechanism of P450 enzymes that accommodate bulky substrates.

Purpose To compare the treatment effects and tolerability of a topical application of mizoribine (MZR) and cyclosporine A (CsA) eye drops (Restasis; Allergan, Inc., Irvine, CA, USA) in a mouse dry eye model. Methods C57BL/6 mice subjected to desiccating stress (DS) were treated with 0.05% MZR in phosphate-buffered saline (PBS) or Restasis eye drops four times a day for 5 days. Untreated mice served as control. Tear secretion, Oregon green dextran staining, and the conjunctival goblet cell quantity were evaluated. The apoptosis and matrix metalloproteinase 9 (MMP-9) in the ocular surface, conjunctival CD4, and T helper-related cytokines were verified. The ocular tolerance of these two drugs was evaluated by observing the mice's behavioral changes. Results Topical administrations of MZR or Restasis both increased tear production, maintained goblet cell density, and improved corneal barrier function. Both MZR and Restasis suppressed the expression of MMP-9 and apoptosis in the ocular surface. Meanwhile, both MZR and Restasis decreased the infiltration of CD4+ T cells, reversed the production of interferon-γ, interleukin (IL)-17A, and IL-13 in conjunctiva under DS. The abovementioned efficacies between these two eye drops were not statistically significant. However, the number of scratching and wiping behaviors in the MZR-treated group was significantly less than in the Restasis-treated group. Conclusions MZR (0.05% in PBS) could be a good competitive product for Restasis because of the comparable treatment effect in dry eye diseases and better ocular tolerability in ocular itch and pain. Translational relevance This study provided an immunosuppressive agent comparable to Restasis for the treatment of dry eye disease.

Background/Aims: Cyclosporine A (CsA) is an immunosuppressant drug that is used during organ transplants. However, its utility is limited by its nephrotoxic potential. This study aimed to investigate whether fluorofenidone (AKF-PD) could provide protection against CsA-induced nephrotoxicity. Methods: Eighty-five male Sprague-Dawley rats were divided into 5 groups: drug solvent, CsA, CsA with AKF-PD (250, 500 mg/kg/day), and CsA with pirfenidone (PFD, 250 mg/kg/day). Tubulointerstitial injury index, extracellular matrix (ECM) deposition, expression of type I and IV collagen, transforming growth factor (TGF)-β1, platelet-derived growth factor (PDGF), Fas ligand (FASL), cleaved-caspase-3, cleaved-poly(ADP-ribose) polymerase (PARP)-1, and the number of transferase-mediated nick end-labeling (TUNEL)-positive renal tubule cells were determined. In addition, levels of TGF-β1, FASL, cleaved-caspase-3, cleaved-PARP-1, and number of annexin V-positive cells were determined in rat proximal tubular epithelial cells (NRK-52E) treated with CsA (20 μmol/L), AKF-PD (400 μg/mL), PFD (400 μg/mL), and GW788388 (5 μmol/L). Results: AKF-PD (250, 500 mg/kg/day) significantly reduced tubulointerstitial injury, ECM deposition, expression of type I and IV collagen, TGF-β1, PDGF, FASL, cleaved-caspase-3, cleaved-PARP-1, and number of TUNEL-positive renal tubule cells in the CsA-treated kidneys. In addition, AKF-PD (400 μg/mL) significantly decreased TGF-β1, FASL, cleaved-caspase-3, and PARP-1 expression in NRK-52E cells and further reduced the number of annexin V-positive cells. Conclusion: AKF-PD protect kidney from fibrosis and apoptosis in CsA-induced kidney injury.

Vinod Burade,1 Rishit Zalawadia,1 Alpesh Patel,1 Abayomi Ogundele2 1Sun Pharmaceutical Industries Limited, Vadodara, Gujarat, India; 2Sun Pharmaceutical Industries, Inc, Princeton, NJ, USACorrespondence: Abayomi OgundeleSun Pharmaceutical Industries, Inc, Princeton, NJ, USATel +1 (609) 720-5389Email Abayomi.Ogundele@sunpharma.comIntroduction: Cyclosporine ophthalmic solution 0.09% (CsA 0.09% sol) is approved to increase tear production in patients with keratoconjunctivitis sicca. This study evaluated the efficacy of CsA 0.09% sol vs cyclosporine ophthalmic emulsion 0.05% (CsA 0.05% eml) vs ciclosporin ophthalmic emulsion 0.1% (CsA 0.1% eml) in a NOD mice model.Methods: Mice were randomized and administered placebo, CsA 0.09% sol twice daily, CsA 0.05% eml twice daily, CsA 0.09% sol once daily, or CsA 0.1% eml once daily in the conjunctival sac of both eyes for 60 days. Tear volume was measured with phenol red threads at baseline and 4 hours after treatment every 15 days. On day 58, the corneal surface was observed under a slit-lamp after staining with 3% lissamine green administered into the inferior lateral conjunctival sac. On day 61, mice were euthanized, globes excised, sliced into 4 μm sections in 3 areas per section, and stained. Total number of stained goblet cell/μm was counted, and the sum per eye was averaged. Lacrimal gland tissues were removed and interleukin (IL) 1-β cytokine levels estimated.Results: Groups comprised 11 mice each, including an untreated normal and untreated diseased control group (7 groups total). CsA 0.09% sol twice daily significantly increased tear volume on day 30, 45, and 60 vs CsA 0.05% eml (P< 0.05, < 0.001, < 0.001, respectively) and vs CsA 0.1% eml on day 60 (P< 0.05); CsA 0.09% sol once daily significantly increased tear volume on day 45 vs CsA 0.05% eml (P< 0.05). Goblet cell density significantly increased with CsA 0.09% sol twice daily vs placebo and NOD control (P< 0.01 both). There was no significant difference in corneal staining and IL-1β levels with CsA 0.09% sol.Conclusion: Sixty-day treatment with CsA 0.09% sol showed comparatively improved preclinical results vs CsA 0.05% eml and CsA 0.1% eml.Keywords: cyclosporine A, goblet cell density, keratoconjunctivitis sicca, preclinical, tear production

Purpose: To compare corneal morphologic changes in vernal keratoconjunctivitis (VKC) patients before and after treatment with topical cyclosporin (CsA).Methods: 23 VKC patients and 40 healthy subjects as controls were considered. All patients were treated with 1%CsA during their active phase. The impact of treatment on quality of life was evaluated administering the QUICK questionnaire. Both eyes were evaluated using the in vivo confocal microscopy (IVCM) before treatment (T0), at the end of treatment in Autumn (T1) and at the recurrence of symptoms in the next season (T2).Results: All VKC patients improved their QUICK scores after CsA treatment. IVCM highlighted corneal microstructural changes at the level of epithelium, sub-basal nerve plexus and stroma in patients affected by VKC and allowed to follow their evolution after topical treatment with CsA.Conclusions: IVCM is a valuable tool to evaluate the dynamic corneal changes in VKC before and after treatment with CsA.

Low aqueous solubility and membrane permeability of some drugs are considered major limitations for their use in clinical practice. Polymeric micelles are one of the potential nano-drug delivery systems that were found to ameliorate the low aqueous solubility of hydrophobic drugs. The main objective of this study was to develop and characterize a novel copolymer based on poly (ethylene glycol) stearate (Myrj™)-block-poly(ε-caprolactone) (Myrj-b-PCL) and evaluate its potential as a nanosystem for ocular delivery of cyclosporine A (CyA). Myrj-b-PCL copolymer with various PCL/Myrj ratios were synthesized via ring-opening bulk polymerization of ε-caprolactone using Myrj (Myrj S40 or Myrj S100), as initiators and stannous octoate as a catalyst. The synthesized copolymers were characterized using 1H NMR, GPC, FTIR, XRD, and DSC. The co-solvent evaporation method was used to prepare CyA-loaded Myrj-b-PCL micelles. The prepared micelles were characterized for their size, polydispersity, and CMC using the dynamic light scattering (DLS) technique. The results from the spectroscopic and thermal analyses confirmed the successful synthesis of the copolymers. Transmission electron microscopy (TEM) images of the prepared micelles showed spherical shapes with diameters in the nano range (